User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13: Difference between revisions

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the second matrix matrix  can be found [http://openwetware.org/wiki/User:Timothy_L._Foley/Notebook/refolding_matrix/2009/10/13/Refolding_Matrix-10-13-2009_amino_acid_4-factor here]
the second matrix matrix  can be found [http://openwetware.org/wiki/User:Timothy_L._Foley/Notebook/refolding_matrix/2009/10/13/Refolding_Matrix-10-13-2009_amino_acids_4-factor here]
or the following matrix with the amino acids as a 4-factor categorical with none, glu, arg, glu+arg
or the following matrix with the amino acids as a 4-factor categorical with none, glu, arg, glu+arg


Revision as of 17:44, 13 October 2009

Refolding Matrix <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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In the beginning there was Jack

getting started here... The background reading I have done that will weigh heavy today: The Pierce Refolding Kit Instructions[1] that detail the 9 buffer components in their kit, which are:

reducing agents
PEG
divalent cations
high [NaCl]
L-arginine
guanidinium chloride (GnCl)

also contained in the kit is an unreleased "Refolding Guide" that comes with the ProMatrix kit an is not available electronically... it would be nice to read this, but C'est la vie!


A paper on High throughput automated refolding [2] that describes a 96 well matrix of buffer conditions they developed for structural genomics projects for Mtb and Viruses.


Finally, Vertex Pharmaceuticals [3] fills the gaps and describes the use statistical software to generate a fractional factorial screen; application of light absorption to measure protein precipitation (λ 390nm); and most importantly statistical analysis of the data (both precipitation and enzyme activity)from the fractional factorial matrix to draw conclusions and further direct optimization.




Designing Our Matrix


The problem with any matrix report, is that they are packed full of misc. chemicals that are either not in house, or prohibitively expensive... it is my experience to find a way to furnish results that are desired first, then ask for $$$ chemicals to improve future experiments second...
This excludes:


all Non Detergent SulfoBetaines (NDSB's) that can or cannot be important
a-cyclodextrin and methyl-B-cyclodextrin (NB: B-cyclodextrin that we have in house is not soluble in H2O.)
fancy detergents (lauryl maltoside)
N-lauryl sarcosine (iFOLD kits)
FoldACEs (iFOLD series 3 kit)
disco reducing agents (BMC: bis-mercaptoacetamide cyclohexane)

additionally,

From Willis' [3] report, buffer pH, detergent, and NDSB 201 were important for some proteins,

So, our matrix will include an expansion of pH, and various detergents around that may or may not help (it's a screen for a reason) additives that are not NDSB's that may help, and some other stuff I like to hypothesize about.

The conditions we will screen follow:

pH/ Buffer
5.5 MES
6.5 MES
7.5 HEPES
8.2 TRIS
9.0 CHES

I replaced BORATE pH 9.5 from [3] with CHES to alleviate complications that will arise during reagent preparation, since borate is only marginally soluble in water, and cannot be prepared as a 10X stock (500 mM).


detergents/ concentration
triton X 100
tween 80
Chaps
none

NB: NONE is a category for all subsequent categories, because it must be included for matrix generation

reducing agents/ concentration
DTT
TCEP
none

additives:
arginine/asparate/none
divalent cations (2 Mg++/ 0.1 Ca++)
GnCl
Ligand
PEG 3350/Glycerol/Sucrose
BSA
NaCl/KCl (high ionic strength)


Why BSA? this is my screen, and a I want to try something wacky.

Stock Solutions

it is important to make sure that your stocks are at concentrations appropriate such that an all positive sample (e.g. one having all possible components) does not have a volume greater than your total volume for the experiment

here is the table I cooked up to figure out what "X" concentration I would need to make everything work, taking into account certain characteristics (e.g. if 20% glycerol will be the final concentration, then i cannot achieve more than a 5X stock solution)

component stock solution relative volume
Buffer 10X 10
detergent 100X 1
amino acids 5X 20
GnCl 10X 10
Ligand 10X 10
PEG 100X 1
Glycerol 5X 20
Salt MIX 10X 10
BSA 10X 10
Reducing agent 100X 1
total 93

NB: I used the totally wicked excel2wiki converter for the table code

so we are set.

component concentrations from [3] units interconversion
Buffer 50 mM
detergent 0.5 mM ~0.06%
amino acids 550 mM
GnCl 550 mM
Ligand 100 uM
PEG 0.06 %
Glycerol 20 %
Salt MIX 264/11 mM Na/K
BSA 10 mg/mL
Reducing agent 5 mM


stock solutions we will need:


stock solution concentration units
Buffer 500 mM
detergent 6 %
amino acids 5500 mM
GnCl 5500 mM
Ligand 1000 uM
PEG 6 %
Glycerol 100 %
Salt MIX 2640/110 mM Na/K
BSA 100 mg/mL
Reducing agent 500 mM

Buffers

Buffer molecular weight stock solution volume mass '
MES 195 500 50 4.875 g
HEPES 238.3 500 50 5.9575 g
TRIS 121.1 500 50 3.0275 g
CHES 207.3 500 50 5.1825 g

salt mix

high salt component stock conc units desired conc vol units volumes '
NaCl 5 M 2.64 50 mL 26.4 mL
KCl 4 M 0.11 50 mL 1.375 mL
water 22.225 mL
low salt
NaCl 5 M 0.264 50 mL 2.64 mL
KCl 4 M 0.011 50 mL 0.1375 mL
water 47.2225 mL

divalent cation mix

divalent cations ' ' stock ' vol tot ' vol '
MgCl2 200 mM 1 M 25 mL 5 mL
CaCl2 10 mM 1 M 25 mL 0.25 mL
total 5.25 mL
vol water 19.75 mL

detergents

detergents stock desired concentration stock (mL) water (mL)
TritonX 25 % 6 % 25 mL 6 19
Tween 80 20 % 6 % 25 mL 7.5 17.5
CHAPS 6 % 25 mL 1.5 g to 25

ligand

ligand stock ' desired ' vol ' stock (uL) ' water '
Ligand A 20 mM 1 mM 10 mL 500 uL 9.5 mL

amino acids

amino acid mw ' stock ' vol ' mass
L-glutamate 147 g/mol 5.5 M 50 mL 40.425
L-arginine 211 g/mol 5.5 M 50 mL 58.025

reducing agents

NB: stocks will be prepared without reducing agents! these should be added fresh every time

reducing agents mw ' stock ' vol ' mass '
DTT 155 g/mol 0.5 M 10 mL 0.775 g
TCEP 250 g/mol 0.5 M 10 mL 1.25 g

Matrix

the first matrix can be found here The above conditions were paired in "Custom Design" of JMP-IN as follows: pH: 5 level Categorical


the second matrix matrix can be found here or the following matrix with the amino acids as a 4-factor categorical with none, glu, arg, glu+arg

buffer # pH detergent amino acids salt crowders divalent cation GnCl BSA Ligand reducing agent Amino acid
1 5.5 MES none none low none 1 0 1 1 none Arg
2 5.5 MES none none high Glycerol 0 1 1 1 TCEP Glu
3 5.5 MES none Glut none none 0 0 0 0 none Arg+Glu
4 5.5 MES none Glut low Glycerol 0 0 0 0 TCEP none
5 5.5 MES TritonX none high none 1 0 0 1 none Glu
6 5.5 MES TritonX Arg low PEG 1 1 1 1 none Arg+Glu
7 5.5 MES TritonX Arg low Glycerol 0 1 0 0 TCEP Arg
8 5.5 MES TritonX Arg high Glycerol 1 1 1 0 none Arg+Glu
9 5.5 MES TritonX Glut none none 1 0 1 0 TCEP Arg+Glu
10 5.5 MES Tween80 none none PEG 0 1 1 1 DTT none
11 5.5 MES Tween80 none none Glycerol 1 1 0 0 DTT Arg
12 5.5 MES Tween80 Arg none PEG 0 0 0 1 TCEP Arg
13 5.5 MES Tween80 Arg low Glycerol 0 0 0 0 none Glu
14 5.5 MES Tween80 Arg high none 1 1 1 1 DTT Arg
15 5.5 MES CHAPS none high Glycerol 0 0 0 0 DTT Arg+Glu
16 5.5 MES CHAPS Arg none none 1 0 0 1 TCEP none
17 5.5 MES CHAPS Glut none PEG 0 1 0 1 none Glu
18 5.5 MES CHAPS Glut low PEG 1 1 1 0 DTT Glu
19 5.5 MES CHAPS Glut high PEG 1 1 1 0 DTT none
20 6.5 MES none none none none 1 1 0 0 none Arg
21 6.5 MES none none low Glycerol 1 0 0 1 DTT none
22 6.5 MES none Arg none PEG 1 0 0 1 DTT none
23 6.5 MES none Glut high none 0 1 1 0 none Arg+Glu
24 6.5 MES none Glut high PEG 0 1 0 0 TCEP Arg+Glu
25 6.5 MES TritonX none low none 0 1 0 0 TCEP none
26 6.5 MES TritonX none high Glycerol 1 1 1 1 DTT Arg
27 6.5 MES TritonX Arg none none 0 1 0 0 none Glu
28 6.5 MES TritonX Arg high PEG 1 0 1 1 DTT Arg+Glu
29 6.5 MES TritonX Glut low Glycerol 1 0 1 1 none Arg
30 6.5 MES Tween80 Arg none none 0 1 1 1 DTT Arg
31 6.5 MES Tween80 Arg none Glycerol 0 0 1 1 none Arg+Glu
32 6.5 MES Tween80 Glut low PEG 1 1 0 0 TCEP Glu
33 6.5 MES Tween80 Glut low Glycerol 1 1 1 1 TCEP none
34 6.5 MES Tween80 Glut high Glycerol 0 1 1 1 none Glu
35 6.5 MES CHAPS none none none 0 0 1 0 TCEP Arg
36 6.5 MES CHAPS none low PEG 0 0 1 0 none Glu
37 6.5 MES CHAPS none low Glycerol 1 0 0 0 TCEP Arg+Glu
38 6.5 MES CHAPS Arg high PEG 0 0 0 1 TCEP none
39 6.5 MES CHAPS Arg high PEG 1 0 0 0 DTT Glu
40 7.5 HEPES none none high none 1 0 0 1 TCEP Glu
41 7.5 HEPES none Arg none Glycerol 0 0 1 0 DTT Glu
42 7.5 HEPES none Glut none Glycerol 1 1 1 1 TCEP Arg+Glu
43 7.5 HEPES none Glut low none 0 0 1 1 DTT none
44 7.5 HEPES none Glut low PEG 0 0 0 1 DTT Arg
45 7.5 HEPES TritonX none none PEG 1 1 1 0 DTT Glu
46 7.5 HEPES TritonX none low none 0 1 0 1 DTT Glu
47 7.5 HEPES TritonX none high PEG 0 0 1 1 none Arg
48 7.5 HEPES TritonX Arg none PEG 0 0 1 0 none none
49 7.5 HEPES TritonX Glut high Glycerol 0 0 0 0 TCEP Glu
50 7.5 HEPES Tween80 none low PEG 1 0 1 1 TCEP Arg+Glu
51 7.5 HEPES Tween80 none high Glycerol 1 0 1 0 TCEP Arg+Glu
52 7.5 HEPES Tween80 Arg high none 1 0 0 0 DTT none
53 7.5 HEPES Tween80 Glut none PEG 1 1 0 0 TCEP Arg+Glu
54 7.5 HEPES Tween80 Glut high none 1 1 0 0 none Arg
55 7.5 HEPES CHAPS none none none 0 1 1 1 none none
56 7.5 HEPES CHAPS Arg none Glycerol 1 1 0 0 none none
57 7.5 HEPES CHAPS Arg low none 0 1 1 0 DTT Arg+Glu
58 7.5 HEPES CHAPS Arg low Glycerol 1 1 0 1 TCEP Arg
59 7.5 HEPES CHAPS Glut high Glycerol 0 1 0 1 none Arg
60 8.2 Tris none none none PEG 0 1 0 0 TCEP Arg
61 8.2 Tris none none none Glycerol 0 1 1 0 none none
62 8.2 Tris none Arg high none 1 1 1 1 TCEP Glu
63 8.2 Tris none Arg high PEG 0 1 0 1 none Arg+Glu
64 8.2 Tris none Arg high PEG 1 0 0 0 DTT Arg
65 8.2 Tris TritonX none low PEG 0 0 0 1 none Arg+Glu
66 8.2 Tris TritonX Glut none PEG 1 1 1 0 TCEP Arg
67 8.2 Tris TritonX Glut none Glycerol 1 0 1 1 TCEP none
68 8.2 Tris TritonX Glut low none 1 1 0 0 DTT none
69 8.2 Tris TritonX Glut high Glycerol 0 0 0 1 DTT none
70 8.2 Tris Tween80 none low none 0 0 1 0 DTT Arg
71 8.2 Tris Tween80 none high none 0 0 0 0 none none
72 8.2 Tris Tween80 Arg low PEG 0 1 1 1 TCEP Glu
73 8.2 Tris Tween80 Arg high none 1 0 1 0 TCEP Arg+Glu
74 8.2 Tris CHAPS none none Glycerol 1 1 0 1 DTT Arg+Glu
75 8.2 Tris CHAPS Arg low Glycerol 0 1 1 0 DTT Glu
76 8.2 Tris CHAPS Glut none none 1 0 1 1 none Glu
77 8.2 Tris CHAPS Glut low Glycerol 1 0 0 1 none Arg
78 9.2 CHES none none none Glycerol 1 0 1 0 DTT Glu
79 9.2 CHES none Arg low none 0 1 0 1 TCEP Arg+Glu
80 9.2 CHES none Arg low PEG 1 0 1 0 none Arg
81 9.2 CHES none Arg low Glycerol 1 1 1 0 none none
82 9.2 CHES none Glut high none 1 1 1 1 DTT Glu
83 9.2 CHES TritonX none high Glycerol 0 1 1 0 TCEP none
84 9.2 CHES TritonX Arg none none 1 0 0 1 TCEP Glu
85 9.2 CHES TritonX Arg high Glycerol 0 1 0 0 DTT Arg
86 9.2 CHES TritonX Glut none PEG 0 1 0 1 DTT Arg+Glu
87 9.2 CHES Tween80 none low PEG 1 1 0 1 none none
88 9.2 CHES Tween80 none high PEG 1 1 0 0 none Glu
89 9.2 CHES Tween80 Arg none Glycerol 1 0 0 1 none Glu
90 9.2 CHES Tween80 Glut none Glycerol 0 0 0 1 DTT Arg+Glu
91 9.2 CHES Tween80 Glut low none 0 0 1 0 DTT Arg+Glu
92 9.2 CHES CHAPS none low none 1 1 0 1 DTT Arg+Glu
93 9.2 CHES CHAPS none high PEG 0 0 1 1 TCEP Arg
94 9.2 CHES CHAPS Arg high none 0 1 1 1 TCEP none
95 9.2 CHES CHAPS Glut none none 0 0 1 0 TCEP Arg
96 9.2 CHES CHAPS Glut high PEG 1 0 1 0 none none

References

  1. http://www.piercenet.com/files/1453as4.pdf

    [Pierce]
  2. Vincentelli R, Canaan S, Campanacci V, Valencia C, Maurin D, Frassinetti F, Scappucini-Calvo L, Bourne Y, Cambillau C, and Bignon C. High-throughput automated refolding screening of inclusion bodies. Protein Sci. 2004 Oct;13(10):2782-92. DOI:10.1110/ps.04806004 | PubMed ID:15388864 | HubMed [Vincentelli]
  3. Willis MS, Hogan JK, Prabhakar P, Liu X, Tsai K, Wei Y, and Fox T. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions. Protein Sci. 2005 Jul;14(7):1818-26. DOI:10.1110/ps.051433205 | PubMed ID:15937284 | HubMed [Willis]
All Medline abstracts: PubMed | HubMed



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